Biophysical investigation of type A PutAs reveals a conserved core oligomeric structure

Korasick, D. A., Singh, H., Pemberton, T. A., Luo, M., Dhatwalia, R., & Tanner, J. J.

The FEBS journal 284.18 (2017): 3029-3049.

Many enzymes form homooligomers, yet the functional significance of self-association is seldom obvious. Herein, we examine the connection between oligomerization and catalytic function for proline utilization A (PutA) enzymes. PutAs are bifunctional enzymes that catalyze both reactions of proline catabolism. Type A PutAs are the smallest members of the family, possessing a minimal domain architecture consisting of N-terminal proline dehydrogenase and C-terminal l-glutamate-γ-semialdehyde dehydrogenase modules. Type A PutAs form domain-swapped dimers, and in one case (Bradyrhizobium japonicum PutA), two of the dimers assemble into a ring-shaped tetramer. Whereas the dimer has a clear role in substrate channeling, the functional significance of the tetramer is unknown. To address this question, we performed structural studies of four-type A PutAs from two clades of the PutA tree. The crystal structure of Bdellovibrio bacteriovorus PutA covalently inactivated by N-propargylglycine revealed a fold and substrate-channeling tunnel similar to other PutAs. Small-angle X-ray scattering (SAXS) and analytical ultracentrifugation indicated that Bdellovibrio PutA is dimeric in solution, in contrast to the prediction from crystal packing of a stable tetrameric assembly. SAXS studies of two other type A PutAs from separate clades also suggested that the dimer predominates in solution. To assess whether the tetramer of B. japonicum PutA is necessary for catalytic function, a hot spot disruption mutant that cleanly produces dimeric protein was generated. The dimeric variant exhibited kinetic parameters similar to the wild-type enzyme. These results implicate the domain‐swapped dimer as the core structural and functional unit of type A PutAs.

Biophysical-investigation-of-type-A-PutAs-reveals-a-conserved-core-oligomeric-structure

Products Used in this Publication

N-propargylglycine (NPPG)

Related Products

Catalog NumberCASProduct NamePrice
B0001-054910113-73-5Gramicidin SInquiry
B0001-1498097161-73-1Thiocholine iodideInquiry
B0001-16063111096-26-7ErythropoietinInquiry
B0001-186805203787-91-1Salcaprozate sodium, 99%Inquiry
B0001-3946391341-23-7Nicotinamide Riboside (Contain ~80% food grade dextrin as stabilizer)Inquiry
Price Inquiry